Kidney function, both under normal conditions and in the setting of injury or disease, is predominantly controlled by the amount, localization and activation state of proteins in specific cells at specific sites and under specific spatial influences that in turn regulate cell differentiation, division, metabolism, morphology, membrane polarization, secretion and transport. Imaging mass cytometry (IMC) takes advantage of laser ionization and time of flight mass spectrometry to simultaneously identify >40 metal ion-conjugated antibodies from tissue sections at 1micron resolution with essentially no background signal. By mixing cell specific antibodies and activation state-specific antibodies in the same cocktail, IMC produces a spatially preserved analysis of the location and activation state of multiple cell types from the same tissue section. We have developed a panel of 21 antibodies for simultaneous kidney section hybridization and IMC analysis, and a machine-learning based analysis pipeline that has allowed us to provide the first quantitative atlas of the number and location of the tubular, endothelial, glomerular and interstitial cells in the normal human kidney (Singh et al., JCI-Insight, in press). We now propose to validate an additional 22-29 antibodies for an expansion of this panel so as to provide information on the immune activation state and resident epithelial and endothelial cell injury, survival and death pathway responses predicted to be important in the injured human kidney. We will first perform in vitro validation of antibodies that detect cell injury/dedifferentiation states, survival pathways (autophagy, unfolded protein response, proliferation), and cell death/senescence pathways (apoptosis, necroptosis, pyroptosis, senescence, Specific Aim 1), as well as immune cell populations and their activation states (Specific Aim 2). We will then perform IMC/Kidney-MAPPS based validation of the composite panel using carefully characterized human kidney biopsies from patients with defined clinical insults and morphologically quantified tubular and endothelial injury (Specific Aim 3). The end product of this proposal will be to provide a validated set of antibodies that can be used to accurately quantify cell injury states and subsequent responses across a spectrum of human kidney diseases. These antibodies can then be used by investigators who perform multiplexed imaging of the kidney, including IMC, IONpath MIBI cytometry, Tissue Cytometry and Repeated Fluorescent Imaging to mechanistically interrogate human AKI.